Structural studies of biological macromolecules always start with producing the molecules in microgram to milligram quantities suitable for single molecule electron microscopy (EM), X-ray crystallography or NMR measurement. With subnanogram amount of biological macromolecules, it is prohibitively difficult to obtain their three-dimensional (3D) structures using the current technology. We propose to develop innovative technology that will overcome the quantity limit and obtain 3D structures from subnanogram quantities of biological complexes using single molecule EM. The key innovation will be to develop affinity-based methods that will specifically enrich low-abundance biological macromolecules onto carbon films and prepare them for single molecule EM and 3D reconstruction. The feasibility of this technology will be examined by introducing specific biological ligands to the surface of thin carbon films and evaluating the selective binding of cognate biological complexes, and by applying a Ni-chelating surface to the enrichment of a His-tagged protein complex (KvAP/Fv) for calculating its 3D structure. To test the application to a low- abundance complex, we will enrich functional human telomerase complex to the surface of modified carbon films and generate its first 3D structure. In summary, successful advancement of the new technology will make it possible to generate structures of many multi-component complexes only available at subnanogram levels. PUBLIC HEALTH RELEVANCE: With the currently available technology, we need to purify macromolecules at microgram to milligram levels before thinking of obtaining their three-dimensional (3D) structures. With subnanogram quantity of materials, it is almost impossible to obtain 3D structures of target molecules. Single particle electron microscopy calculates 3D structures from thousands of images of individual molecules. With millions of molecular images, it now becomes achievable to generate structures at subnanometer to atomic resolutions. This means that it is feasible to obtain structures with subnanogram materials once we know how to image millions of molecules. This proposal is aimed at developing new technology that will enable us to take images of millions of molecules from subnanogram biological material. It will use nano-scale chemical reactions to anchor specific ligands onto a substrate surface (inert carbon film), and bind selectively low-abundance biological macromolecules that can only be pre-purified to ~0.1 nanogram level. These molecules will then be prepared for EM imaging and reconstruction of their structures. The new technology will expand the structural study to many important macromolecular complexes that are difficult to produce in large quantities but play pivotal roles in various cellular functions.